Bottom Line:
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs).MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs.A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8.

ABSTRACTMCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.

f1: MCM8-9 is required for RPA binding to DSB sites.(a) Immunoblots of lysates from U2OS cells transfected with indicated siRNAs, 4 h after addition of cisplatin. (b) Decrease of RPA70 foci on depletion of MCM8 or MCM9. RPA70 immunofluorescence images shown at left and cells having over 20 foci of RPA70 were counted as positive cells on the right. Scale bar, 10 μm. ***P<0.005; Student's t-test. (c) ChIP assay in HeLa DR13-9 cells. Fold signal of RPA70 at I-SceI cut site relative to control site 2 kb upstream of cut site in cell treated with indicated siRNAs. *P<0.05; Student's t-test. (d) Immunoblot of U2OS cells stably transfected with empty vector (EV) or plasmid expressing siMCM8 resistant, Flag-MCM8r, 48 h after transfection of siRNAs. (e) Restoration of RPA foci formation by siMCM8-resistant Flag-MCM8. Quantification of RPA70 or γH2AX foci-positive cells as in Fig. 1b. ***P<0.005; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole.

Mentions:
Deficiency of MCM8 or MCM9 impairs the recruitment of Rad51 on chromatin after DNA damage1517. DNA resection by the MRN complex, CtIP, EXO1 and DNA2, produces the ssDNA that has to be coated by RPA before subsequent Rad51 assembly1920. We thus measured the accumulation of RPA at DNA damage sites in MCM8- or MCM9-depleted cells. As reported previously, depletion of MCM8 decreases MCM9, but depletion of MCM9 does not deplete MCM8 (Fig. 1a). Even though levels of RPA70 were unchanged, there was a decrease in cisplatin-induced RPA foci formation on MCM8 or MCM9 depletion (Fig. 1a,b). HeLa DR13-9 cells contain a single I-SceI cut site integrated into their genome that is repaired by HR after cleavage by the I-SceI21. Although the expression level of I-SceI was not diminished in MCM8- or MCM9-depleted cells (Supplementary Fig. 1), RPA binding to the I-SceI cut site was decreased in cells depleted of MCM8 or MCM9 compared with control short interfering RNA (siRNA; siGL2)-transfected cells (Fig. 1c). To eliminate the possibility of off-target effects of siRNAs, we rescued cisplatin-induced RPA foci formation in siMCM8-transfected cells by stably expressing siRNA-resistant Flag-tagged MCM8 (Flag-MCM8r; Fig. 1d,e). H2AX phosphorylation on S139, and by inference the number of DNA breaks, was not affected by MCM8-9 depletion, suggesting that MCM8-9 specifically affected a step after DNA break formation.

f1: MCM8-9 is required for RPA binding to DSB sites.(a) Immunoblots of lysates from U2OS cells transfected with indicated siRNAs, 4 h after addition of cisplatin. (b) Decrease of RPA70 foci on depletion of MCM8 or MCM9. RPA70 immunofluorescence images shown at left and cells having over 20 foci of RPA70 were counted as positive cells on the right. Scale bar, 10 μm. ***P<0.005; Student's t-test. (c) ChIP assay in HeLa DR13-9 cells. Fold signal of RPA70 at I-SceI cut site relative to control site 2 kb upstream of cut site in cell treated with indicated siRNAs. *P<0.05; Student's t-test. (d) Immunoblot of U2OS cells stably transfected with empty vector (EV) or plasmid expressing siMCM8 resistant, Flag-MCM8r, 48 h after transfection of siRNAs. (e) Restoration of RPA foci formation by siMCM8-resistant Flag-MCM8. Quantification of RPA70 or γH2AX foci-positive cells as in Fig. 1b. ***P<0.005; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole.

Mentions:
Deficiency of MCM8 or MCM9 impairs the recruitment of Rad51 on chromatin after DNA damage1517. DNA resection by the MRN complex, CtIP, EXO1 and DNA2, produces the ssDNA that has to be coated by RPA before subsequent Rad51 assembly1920. We thus measured the accumulation of RPA at DNA damage sites in MCM8- or MCM9-depleted cells. As reported previously, depletion of MCM8 decreases MCM9, but depletion of MCM9 does not deplete MCM8 (Fig. 1a). Even though levels of RPA70 were unchanged, there was a decrease in cisplatin-induced RPA foci formation on MCM8 or MCM9 depletion (Fig. 1a,b). HeLa DR13-9 cells contain a single I-SceI cut site integrated into their genome that is repaired by HR after cleavage by the I-SceI21. Although the expression level of I-SceI was not diminished in MCM8- or MCM9-depleted cells (Supplementary Fig. 1), RPA binding to the I-SceI cut site was decreased in cells depleted of MCM8 or MCM9 compared with control short interfering RNA (siRNA; siGL2)-transfected cells (Fig. 1c). To eliminate the possibility of off-target effects of siRNAs, we rescued cisplatin-induced RPA foci formation in siMCM8-transfected cells by stably expressing siRNA-resistant Flag-tagged MCM8 (Flag-MCM8r; Fig. 1d,e). H2AX phosphorylation on S139, and by inference the number of DNA breaks, was not affected by MCM8-9 depletion, suggesting that MCM8-9 specifically affected a step after DNA break formation.

Bottom Line:
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs).MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs.A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8.

ABSTRACTMCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.